Numerous techniques and devices are known and available for imaging structures or objects within opaque or turbid mediums, such as biological tissues. Various examples and a description of such techniques are described in related applications, Ser. No. 07/722,823 (filed Jun. 29, 1992), entitled "Method and Apparatus for Imaging a Physical Parameter in Turbid Media Using Diffusive Waves," and Ser. No. 07/789,517 (filed Nov. 8, 1991), entitled "Multidimensional Imaging Using a Single Point Detector for a Phase Encoded Modulated Optical Carrier," the respective contents of which are incorporated herein by reference. Various principles embodied in such techniques are further set forth in Knuttel et al., Improvement of Spatial Resolution in Reflectance Near-Infrared Imaging by Laser-Beam Interference, SPIE--The International Society of Optical Enginerring: Time-Resolved Laser Spectroscopy in Biochemistry III, vol. 1640 (January 1992), Feddersen et at., Digital Parallel Acquisition in Frequency Domain Fluorometry, 60 Rev. Sci. Instrum. 2929 (1989) (heterodyne down beating to process signals from diffuse photon density waves), and J. Fiskin et al., Diffusion of Intensity Modulated Near -Infrared Light in Turbid Media, Proc. SPIE, Time-Resolved Spectroscopy and Imaging of Tissues, 1431, 122-135 (1991) (describing the properties and mathematic models for diffuse photon density waves propagating through turbid medium), the respective contents of each of which also are incorporated herein by reference.
The previous methods for spatial localization and imaging through highly scattering media have extended near-infrared spectroscopy by introducing time-resolved techniques. One objective of such imaging is to spatially localize or image optical parameters, like absorption, which is often times different in healthy and cancerous or ischemic tissues. A discussion of these prior techniques and their underlying principles is set forth below.
A diffuse photon density wave is produced when photons from a source of light penetrate a turbid media with a thicknesses exceeding about 10 times the mean-free path of the photon and are multiply scattered therein. The photons propagating through the medium in the diffuse photon density wave are scattered and absorbed such that any coherence in the individual wavelengths of the photons is lost. However, the photon density wave (i.e., a wave propagating through the medium representing the density of the photons) moves through the medium with a coherent wave front. Previous techniques have attempted to obtain information about the medium and objects located in the medium by exploiting the information contained in the diffuse photon density waves, since the propagation depends on the properties of the medium. Various techniques have been proposed both in reflectance-mode and transmittance-mode for optically imaging (i.e., examining optical properties) in biological tissues thicker than a few millimeters. The photon density wave, particularly in reflectance-mode imaging, contributes overwhelmingly to the detected signal because the number of re-emitted (quasi-) ballistic photons (i.e., photons not scattered or absorbed) is extremely low.
Prior art techniques, such as those described in the above-mentioned related applications and publications, have increased the amount of information obtainable from the diffuse wave by the use of intensity (i.e., amplitude) modulation and phase modulation of the source of imaging electromagnetic radiation (e.g., a laser beam). In diffusive wave optics, the wavelength and attenuation of an intensity-modulated photon-density wave are complex functions of modulation-frequency and absorption. Nonetheless, at a given modulation frequency, the photon-density wave travels with constant phase velocity in a homogeneous tissue, which implies that its phase-front maintains coherence. The wavelength of a diffusive photon density wave increases as the absorption increases, because long photon paths become less likely. Attenuation of the diffusive photon density wave is exponential as it propagates through a turbid medium.
Two types of detection systems are used for optical imaging. The term "reflectance mode" refers to the situation where the detector is located on the same side of the medium as the source of light and detects light returning from the medium. The term "transmission mode" detection is used when the detector is placed on the opposite side of the medium detecting light traveling through the medium. The reflectance mode has, compared to the transmission mode, the advantage that very thick (in vivo) objects can be probed, as long as the region of interest is not greater than a few centimeters deep. In some conventional source/detector arrangement, photons returning from regions close to the surface tend to overwhelm those returning from deeper regions. Therefore, the acquired signal is most sensitive to absorbing bodies or emitting probes in the medium located in superficial regions close to the source or the detector.
One objective of more recent prior art imaging devices is to eliminate the problems associated with photons returning from near the surface which tend to overwhelm the detector. In order to enhance spatial localization (imaging) of absorbing or fluorescent bodies deep inside a turbid medium, interfering photon density waves have been produced by various dual laser-source configurations and detected by a single detector or a gated, intensified CCD (ICCD) camera. Utilizing these techniques, two problems associated with traditional reflectance-mode imaging have been ameliorated: (a) the dominance of surface signals and (b) the poor sensitivity to changes in the position of an absorbing body located deep within the medium.
In order to better understand the instant invention an illustration of such a prior art imaging technique is provided. FIG. 1 shows a frequency-domain spectrometer according a prior technique which is configured to image an object embedded in tissue with two interfering diffusive photon density waves and to detect the magnitude and phase of the re-emitted light. In FIG. 1, the spectrometer has a detector 60. The detector is based on either the photo-multiplier tube (PMT) 61a or a gated, intensified CCD (ICCD) camera 61b. A cw mode-locked Nd:YAG laser 101 (Spectra Physics, Model 3400) pumps a cavity dumped dye laser 102 (Model 3510/3295) to produce pulses of .about.10 ps duration at a repetition rate of 4.1 MHz. The average power is about 100 Mw at an optical wavelength of 605 nm and 150 Mw at 575 nm. A small portion of the beam is directed onto a photodiode or onto a few outer pixels of the ICCD camera via mirrors M.sub.1, M.sub.2, beam-splitter Bs.sub.1, and mirrors M.sub.3, and M.sub. 4 to provide a reference signal. Another beam-splitter BS.sub.2 divides the main part of the intensity-modulated beam into two beams 400 and 500. When the device is operated in an asymmetrical mode, beam 400 and beam 500a are applied to the same side of the turbid medium 20 as compared to the detector 60. In the symmetrical configuration, beam 400 and beam 500b are centered around the detector. The two types of configurations are accomplished by inserting and removing mirror M1b. The path length (and therefor phase) of beam 500 can be adjusted by a prism 103 mounted on a rail. The intensity ratio of the two beams is varied by attenuating beam 400 with a variable neutral-density filter 104. The beams 400 and 500a or 500b are directed to a turbid medium 20 having an object 30 therein. The light exiting the object is focused via a relay lens 62 through aperture A and onto detector 60. When using the detector 61a, the PMT signal, as well as the PD signal, are mixed down externally by superheterodyning via an intermediate frequency of 200 kHz. The filtered audio-frequency signal (40 Hz) is acquired with an A/D converter (Keithley). When using the detector including ICCD 61b, a high-frequency sine wave is applied to the intensifier of the ICCD, operating in a fairly linear part of its characteristic curve, to mix the signals internally; the resulting 1 Hz signal is acquired by a frame grabber (Data Translation). The neutral-density filter 105 adapts the light-intensity range to the dynamic range of the particular detection system.
The apparatus of FIG. 1 enables two types of imaging to be preformed, both with the aid of two interfering photon density waves. The apparatus may be used to detect an absorbing body deep inside the turbid medium 20 by desensitizing the region close to the surface. This is accomplished using the asymmetric laser-beam 400 and 500a configuration and the single-detector 61a arrangement. The second type of imaging spatially localizes (images) fluorescent objects inside the medium (20). This is accomplished with the symmetrical laser beam 400 and 500b configuration. The excitation of a fluorescent object may be determined by the phase and magnitude of the photon density at a given location which defined the destructive interference pattern. Light at an excitation wavelength (e.g., 575 nm) is blocked by an optical filter 63 so that transmitted light within a narrow band of wavelength (e.g., 610 nm) around the known emission peak of the fluorescent object is provided to the ICCD camera detection configuration 61b.
For both types of imaging, destructive interference at a given point in the medium is accomplished by adjusting the magnitude and phase of both laser beams until the AC magnitude at that point becomes vanishingly small. This null condition indicates that the AC magnitudes of the diffusing photon density waves produced by the two incident sources are equal in magnitude and 180.degree. out of phase at this point. Imaging data may also be collected utilizing the characteristic of constructive interference. This is accomplished by shifting the phase of beam 500 via prism 103 by 180.degree..
Imaging may be performed by the described apparatus in accordance with the following principles. A prediction of the effect of the position of an absorbing body on the phase and magnitude of the detected signal at a particular frequency can be mathematically approximated. For detecting absorption, the asymmetric laser-beam illumination beam 400 and 500a and signal-point detection apparatus 61a is used. Similarly, a prediction of the illumination pattern in a turbid medium produced by symmetrically positioned laser beams can be calculated using an analytical expression. An object 30 inside the turbid medium 20 is imaged by reference to predicted properties of the diffuse photon density wave in a homogeneous turbid medium. The detected results are interpreted in accordance with the expected characteristics of the diffuse waves in such a turbid medium.
While several mathematical models, including Gaussian-convolution, Monte-Carlo, finite-element, and finite-difference methods, are applicable to all kinds of body/background geometries characterized by a wide range of scattering and absorption coefficients, the principal of time-dependent diffusion of photons as detected by the above apparatus is sufficiently understood using the finite-difference method.
The diffuse photon density .PHI.(r,t) at a given point r and time t can be determined using the time-dependent diffusion equation, ##EQU1## where S(r,t) represents the intensity of the photon source. The speed of light in the medium is c.sub.n =c/n, where n is the refractive index of the medium and c is the vacuum light speed. The term .mu..sub.ab +.mu..sub.a (r) is the total absorption coefficient which is given by the sum of the background absorption coefficient, .mu..sub.ab, and a space-dependent absorption coefficient representing that of the absorbing body, .mu..sub.a (r). The diffusion coefficient is defined in terms of .mu..sub.ab and transport-corrected scattering coefficient, .mu..sub.s ', as ##EQU2## In a turbid medium of anisotropic scatterers, .mu..sub.s ' can be much less than the isotropic scattering coefficient .mu..sub.s. The anisotropy parameters of most biological tissues are between 0.8 and 0.97, yielding values of .mu..sub.s ' typically a factor of 5 to 30 times less than .mu..sub.s.
In accordance with the above calculations, and in particular the diffuse equation (1), the data obtained using the above apparatus can be interpreted. A more detailed explanation of the calculations and characteristics of an intensity modulated diffuse photon density wave propagation is provided in Fiskin et al. mentioned above. FIGS. 2a)-2d) show plots of the intensity and phase data which are produced when an absorbing body is positioned at various locations in the depth and lateral directions. Such imaging is performed using the asymmetric illumination conditions and a single detector (PMT) depicted in FIG. 1. By way of example, these graphs illustrate imaging using a modulation-frequency of 410 MHz where the absorption coefficient, .mu..sub.a1, of the object to be detected is 17.5 times higher than the background absorption under simulated conditions. The spatial zero point for this plot is referenced to the spot where the light exits the medium 20 through the detector 60 aperture. Magnitudes, when using either destructive and constructive interference are normalized to the maximum signal recorded under constructive interference conditions.
As can be seen in FIGS. 2a)-2d), when using this apparatus, the sensitivity is reduced close to the surface. This is because destructive interference occurs in this region. With increasing depth of the absorbing body, the AC magnitude reaches a maximum and finally drops again. Therefore, the region of greatest sensitivity to the absorbing body is crudely localized. The most sensitive region (maximum AC magnitude) under the above conditions is about 13 mm deep for lateral positions close to the detector. The sensitivity maximum shifts closer to the surface as the location of the absorber shifts to positions near the entry point of beam 500a in FIG. 1. In FIG. 2b), a large phase change vs. depth is apparent. Contrast this with the data obtained under constructive-interference conditions shown in FIGS. 2c) and 2d). The magnitude decrease is plotted relative to the maximum signal at the deepest body location to allow comparison with the destructive case. As seen in FIG. 2d), the phase gradient measured under constructive-interference conditions is much smaller than that measured under destructive-interference conditions.
It can further be realized that as the absorption coefficient of an object to be detected changes, the AC magnitude and the phase signals of the detected body vary as shown in FIG. 3. FIGS. 3a and 3b show the relationship between the absorption coefficient and the AC magnitude and phase signals, respectively. The magnitude and phase results are plotted vs. object position in the depth dimension for an object which is laterally displace 20 mm from the detector (located between beam 400 and detector 60 aperture). The magnitude plots have similar shapes, but the change induced by the low absorption body is substantially less than that induced by the high absorption body. While magnitude signals are sensitive to the absorption coefficient of the body, the phase signal is relatively invariant with respect to this parameter.
According to the above apparatus, destructive and constructive interference of diffusive photon density waves may be used to accomplishes two useful functions central to the problem of locating absorbing bodies and imaging fluorescent objects in a turbid medium. First, with respect to the localization of an absorbing body, the region of maximum sensitivity can be shifted deeper into the medium using an asymmetrical laser-beam arrangement. This allows an immediate crude localization. Second, spatially selective excitation of a fluorescent probe in a scattering volume can be achieved by adjusting the phases and magnitudes of two laser beams incident on the surface to establish destructive interference in desired regions. The use of an ICCD camera facilities the simultaneous acquisition of the phase and magnitude signals needed to reconstruct high-resolution images.
Other ways of employing diffusive-wave interference for localization/imaging have been suggested. In FIG. 4, for example, the shapes of the null regions for two different incident beam-intensity ratios and phase differences are depicted which could be directly exploited to localize fluorescent probes in two dimensions. Such contour plots can be calculated using the diffuse wave calculations described above. The magnitude ratios plotted in FIGS. 4a) and 4c) demonstrate that constructive and destructive interference patterns differ significantly only in the vicinity of the null point. It has been considered that by combining the calculated destructive/constructive ratios (FIGS. 4b) and 4d)) obtained for different source positions, a well-defined region of localization can be established, as illustrated in FIG. 4e). Since the signal in the null region is almost zero (noise floor) the position of the peak indicates the region of complete destructive interference very well. While these imaging predictions show the potential for developing multiple-beam systems in imaging applications, modeled after "beam steering" techniques employed in phased-array radar, it is extremely cumbersome to calculate the interference patterns and exploit these possibilities. An even more challenging task of imaging embedded absorbers is believed possible by the above technique by ratioing phase and magnitude signals obtained under different interference conditions to reduce the effects of shallow-penetrating photons that do not interact with the absorbers. Such techniques are still complicated due to the extreme complexity that is associated with the propagation (due to scattering and absorption by the medium) of the photon density waves.
A common problem associated with each of the above techniques is that they involve interpreting complicated diffuse wave interference patterns and output, making it extremely difficult to isolate the portions of the actual output which are due only to the object in question (i.e., filter out the portions of the output which is not due to the object to be imaged and therefore does not contain any useful information). Further, since the useful information is carried in the same domain as the unwanted information the equipment must have extreme precision in order to maintain an acceptable signal to noise ratio. As a result of these problems, use of such apparatus for imaging purposes has been substantially limited.